Antibiotic resistance is widespread and increasing, and there is an urgent clinical need for new antimicrobial targets and therapies. The most attractive new targets are those in pathways where inhibitors have an established history of clinical success. We therefore focus on identifying new vulnerabilities in the bacterial cell wall/peptidoglycan (PG) assembly process, the primary target of one of our most successful antibiotic classes, the ?-lactams. Our goal is to use chemical screens to identify small molecules with broad-spectrum antibiotic activity and in vivo efficacy. To do so, we will develop smart chemical screens that incorporate pathway-specificity in their readout, taking advantage of knowledge gained from our basic mechanistic studies of PG biogenesis in E. coli In Aim 1, we exploit our discovery of specialized growth conditions that render a highly conserved PG-synthesizing complex called the Rod system simultaneously non-essential and toxic. We will identify inhibitors of this normally essential complex by screening for growth-promoting molecules. The positive readout of this chemical suppressor screen is a tremendous advantage as it automatically eliminates non-specific cytotoxic compounds. Aim 2 will focus on characterizing an enzymatic activity that has long been thought to be essential for PG synthesis but has eluded researchers for decades. This activity is part ofthe Rod system. Our results will therefore both define a highly attractive new antibiotic target and aid in the characterization of the Rod system inhibitors identified in Aim 1. Aim 3 describes a new approach to antibiotic discovery. Rather than focusing on small molecules that inhibit essential processes as is traditionally done, we will use our knowledge of the regulatory pathways controlling cell wall degrading enzymes to implement a pathway-directed screen for compounds that aberrantly trigger their activation and induce lysis. Aim 4 is dedicated to using a novel high-throughput genetic approach in E. coli and S. aureus to identify new opportunities for applying pathway-directed screens for antibiotics. The most promising compounds identified in our screens will be optimized in collaboration with the Center's Discovery and Translational Service Core. Those with broad-spectrum activity, high potency (MIC <25 pM), and low cytotoxicity (toxic dose >10x MIC) will be further tested for in vivo efficacy by the core. Industry partners will be sought to move effective molecules into a pipeline for the generation of new therapies to treat infections caused by antibiotic-resistant bacteria.